Comparison of the effects of oxidative and inflammatory stresses on rat chondrocyte senescence

Osteoarthritis (OA) is an age-related degenerative joint disease that causes progressive cartilage loss. Chondrocyte senescence is a fundamental mechanism that contributes to the imbalance of matrix homeostasis in OA by inducing senescence-associated secretory phenotype (SASP). Although OA chondrocytes are mainly exposed to oxidative and inflammatory stresses, the role of these individual stresses in chondrocyte senescence remains unclear. In this study, we compared the effects of these stresses on the senescence of rat chondrocytes. Rat chondrocytes were treated with H2O2 and a combination of IL-1β and TNF-α (IL/TNF) to compare their in vitro effect on senescent phenotypes. For in vivo evaluation, H2O2 and IL/TNF were injected into rat knee joints for 4 weeks. The in vitro results showed that H2O2 treatment increased reactive oxygen species, γ-H2AX, and p21 levels, stopped cell proliferation, and decreased glycosaminoglycan (GAG)-producing ability. In contrast, IL/TNF increased the expression of p16 and SASP factors, resulting in increased GAG degradation. Intraarticular injections of H2O2 did not cause any changes in senescent markers; however, IL/TNF injections reduced safranin O staining and increased the proportion of p16- and SASP factor-positive chondrocytes. Our results indicate that oxidative and inflammatory stresses have significantly different effects on the senescence of rat chondrocytes.

Oxidative stress inhibited the proliferation of rat chondrocytes. The effect of oxidative and inflammatory stresses on cell proliferation was examined via cell counting (Fig. 3A). No differences were observed in terms of cell number at days 2 and 5 between the control and IL/TNF groups (Fig. 3B). By contrast, H 2 O 2 -treated cells hardly proliferated, and the number of cells at days 2 and 5 were significantly lower in H 2 O 2 group than in the control group. Cell cycle analysis using PI revealed that the proportion of cells in G2/M phase was higher in H 2 O 2 group than in the control group (Fig. 3C). We evaluated the expression of cell cycle-related proteins by western blot analysis (Fig. 3D). The expression of p16 was significantly decreased in the H 2 O 2 group and significantly increased in the IL/TNF group compared with the control group (Fig. 3E). Conversely, the expression of p21 was significantly increased in the H 2 O 2 group and decreased in the IL/TNF group. There was no difference in the expression of pRb.
Inflammatory stress increased the expression of SASP factors. The mRNA expression of SASP factors (MMP-13, ADAMTS-5, MCP-1, and IL-6) was compared by qPCR. Inflammatory stress significantly enhanced the gene expression of MMP-13, ADAMTS-5, and MCP-1, but not of IL-6. No difference was observed in the expression of any of these genes between the H 2 O 2 group and the control group (Fig. 4A). We examined the protein expression of SASP factors using western blots (Fig. 4B). The protein expression of cleaved MMP-13 was significantly higher in the IL/TNF group than in the control group, while that of the other genes was unchanged (Fig. 4C). Oxidative stress treatment did not affect the expression level of SASP factors except for decreasing pro MMP-13 expression.
Oxidative stress impaired ECM-producing ability. We next generated chondrogenic spheroids from H 2 O 2 − and IL/TNF-treated chondrocytes to evaluate the effect of oxidative and inflammatory stresses on ECMproducing ability (Fig. 5A). Spheroids of all groups were strongly stained with safranin O (Fig. 5B). The intensity of type II collagen staining was equivalent in all groups. Greater type I collagen expression was observed in the H 2 O 2 and IL/TNF group than in the control group, and spheroids from H 2 O 2 -treated cells showed significantly lower GAG and DNA amounts and GAG/DNA ratio than those in untreated cells (Fig. 5C) To induce inflammatory stress, the cells were cultured in the growth medium supplemented with 10 ng/mL IL-1β and 10 ng/mL TNF-α (IL/TNF group). We performed reactive oxygen species (ROS) assay on day 0, cell proliferation and cell cycle assay on day 2, and senescence-associated β galactosidase (SA-β-gal) staining, γ-H2AX immunostaining, cell proliferation assay, western blot, and quantitative real-time PCR (qPCR) on day 5. www.nature.com/scientificreports/ was significantly lower in the IL/TNF group than in the control group; however, no difference was observed in the DNA content and GAG/DNA ratio.
Inflammatory stress increased the degradation of ECM. The effect of oxidative and inflammatory stresses on cartilage ECM degradation was investigated by treating chondrogenic spheroids with H 2 O 2 and IL/ TNF (Fig. 6A). There were no obvious changes in the intensity of safranin O and type ll collagen staining among 3 groups (Fig. 6B). Spheroids treated with IL/TNF showed lower type I collagen expression than those in the control group. Furthermore, biochemical analysis revealed that the treatment of spheroids with IL/TNF significantly decreased the GAG amount (Fig. 6C). GAG/DNA ratios tended to be lower in IL/TNF group than in the control group. On the other hand, H 2 O 2 -treatment decreased the DNA amount but not the GAG amount and GAG/DNA ratio.

Inflammatory stress increased the expression of p16 and SASP factors in vivo. H 2 O 2 and IL/
TNF were injected into rat knee joints to investigate the effects of oxidative and inflammatory stresses in vivo (Fig. 7A). In the control and H 2 O 2 groups, the tibial articular cartilage was strongly stained with safranin O (Fig. 7B). However, IL/TNF injection reduced the staining of the cartilage, specifically on its surface. The Osteoarthritis Research Society International (OARSI) score was significantly higher in the IL/TNF group (Fig. 7C). The expression of senescence markers was also examined by immunostaining (Fig. 8A). The percentage of cells positive for p16, MMP-13, and MCP-1 was significantly higher in IL/TNF group than in the control group (Fig. 8B). ADAMTS-5 positive cells tended to be increased in IL/TNF group. H 2 O 2 injection did not affect the expression of any evaluated markers.

Discussion
Oxidative and inflammatory stresses increased the proportion of SA-β-gal positive cells in this study, indicating that both stresses induced the senescence of rat chondrocytes. The stronger staining intensity in the H 2 O 2 group suggested that oxidative stress caused a greater expression of β-galactosidase than was induced by inflammatory stress. β-galactosidase is a lysosomal hydrolase that cleaves the terminal β-d-galactose residue, and its activity is upregulated in cells with senescent features accumulated in aged and diseased organs 15 . SA-β-gal activity is reported to increase in senescent chondrocytes 16 and is now widely used as a biomarker for senescent cells across species and cell origins. We found that oxidative stress increased the amount of intracellular ROS and expression of γ-H2AX which is present at DNA double-strand break sites 17 , while inflammatory stress did not. Elevated levels of ROS have been reported to impair the repair mechanism of damaged DNA in chondrocytes 18 . These findings suggest that oxidative stress induced DNA damage via ROS in rat chondrocytes, and inflammatory stress does not induce ROS production.   www.nature.com/scientificreports/ As shown in the proliferation and cell cycle assays, oxidative stress completely stopped cell division with G2/M arrest and increased p21 expression. Since ROS/p53/p21 pathway is well known to inhibit cell growth 19,20 , increased ROS levels in the present study would upregulate the expression of p21 to stop chondrocyte proliferation. Bunz et al. reported that increased p21 expression induced G2/M arrest in human colorectal cancer cell lines 21 , which is in line with our results. On the other hand, inflammatory stress increased p16 expression but did not alter the cell cycle or proliferation ability. IL-1β and TNF-α are known to induce p16 expression in rat chondrocytes and human chondrocyte cell lines, respectively 13,14 . Although p16 is generally considered to inhibit cell cycle progression 22,23 , Diekman et al. found that p16 expression was not associated with proliferation arrest in rat chondrocytes 24 , a result that is echoed in our findings. Thus, in rat chondrocytes, increased p16 levels do not always reflect decreased proliferative ability. IL-1β and TNF-α have been reported to activate NFκB and MAPK and stimulate the production of these SASP factors [25][26][27] . On the other hand, western blot analysis showed that protein expression of MMP-13 was upregulated in the IL/TNF group but that of ADAMTS-5 and MCP-1 was not. As we used cell lysates in western blots, intracellular rather than extracellular protein levels were evaluated. ELISA might have been desirable for detecting   www.nature.com/scientificreports/ that IL/TNF treatment did not affect GAG-producing ability; instead, it promoted GAG degradation. IL-1β and TNF-α can induce the production of extracellular proteases such as MMP-13 and ADAMTS-5. It is suggested that these SASP factors are responsible for the degradation of GAGs in this study. Taken together, the effects of oxidative and inflammatory stress on GAG production and degradation differ from each other. The injection of IL/TNF into rat knee joints decreased the intensity of safranin O staining in tibial cartilage, especially on the cartilage surface, and increased the OARSI score. In addition, the proportion of cells positive for  www.nature.com/scientificreports/ p16, MMP-13, ADAMTS-5, and MCP-1 increased in the IL/TNF group. These data are consistent with in vitro results that inflammatory stress induced senescent phenotypes, such as the increased expression of p16 and SASP factors and GAG degradation. Increased p16 expression in chondrocytes has been reported to induce SASP 32 but Diekman et al. recently found that p16 is just a biomarker, and not an inducer, for SASP in chondrocytes 24 .
There is no consensus on the causal relationship between p16 and SASP factors. In contrast, H 2 O 2 injection did not affect the expression of any senescent markers, including p21, whose expression was upregulated in vitro. This indicates that oxidative stress alone cannot induce chondrocyte senescent phenotypes in vivo. We injected 8 mM H 2 O 2 , which was 20 times higher than the concentration used in vitro. Although a higher concentration of H 2 O 2 may allow detection of changes in cartilage structures and senescent markers, it should have been noted that strong oxidative stress induces cell death instead of cellular www.nature.com/scientificreports/ senescence 33,34 . Another possibility is that injected H 2 O 2 did not reach the chondrocytes. However, evaluation of the in vivo ROS distribution using tissue sections is difficult due to the rapid diffusion of ROS. SASP is the most important senescence phenotype in OA progression because it triggers an imbalance in ECM production and degradation. Our findings suggest that inflammatory stress, rather than oxidative stress, is the dominant driver of SASP in OA. The selective elimination of senescent chondrocytes is a promising therapeutic approach for OA 35,36 . However, this approach targets the cells that already undergo senescence and therefore cannot prevent the cells from senescence itself. Regulation of inflammation stress, such as IL-1β and TNF-α signaling will be critical for inhibiting cellular senescence, particularly the acquisition of SASP.
In conclusion, oxidative stress induced DNA damage, increased p21 expression, arrested cell proliferation, and decreased GAG-producing ability in vitro, but caused no changes in senescent phenotypes in vivo. In contrast, inflammatory stress increased the expression of p16 and SASP factors and GAG degradation, both in vitro and in vivo. Our results demonstrated that the effects of oxidative and inflammatory stresses on the senescent phenotypes of rat chondrocytes differed significantly.

Rat chondrocyte isolation.
All experiments and methods were performed in accordance with relevant guidelines and regulations. All animal care and experimental protocols were conducted in accordance with the ARRIVE guidelines and approved by the Animal Committee of Tokyo Medical and Dental University (approval number A2022-084A). Two 12-week-old male Lewis rats were obtained from the Sankyo Labo Service Corporation (Tokyo, Japan). Articular cartilage was harvested from knee joints and digested with 3 mg/mL Clostridium histolyticum-derived collagenase type V (C9263; Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C for 2 h. The cells were filtered through a 70 μm cell strainer (542070; Greiner Bio-one GmbH, Kremsmuenster, Austria) and plated at a density of 5000 cells/cm 2 in a growth medium consisting of Dulbecco's Modified Eagles Medium (DMEM, D6046; Sigma-Aldrich), 10% fetal bovine serum (Lot no. 42F1562K; Thermo Fisher Scientific, Waltham, MA, USA), and 1% antibiotic-antimycotic (15240062; Thermo Fisher Scientific). After 10 days of cultivation, the cells were detached with 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA, 25200-072; Thermo Fisher Scientific) and cryopreserved with a cell banker, 1plus (CB021; Zenoaq, Fukushima, Japan), for future use.

Induction of oxidative and inflammatory stress in chondrocytes.
To induce oxidative stress, rat chondrocytes at passage 2 were treated with 400 μM H 2 O 2 (080-01186; Wako, Tokyo, Japan) for 2 h and then cultured in the growth medium without H 2 O 2 for 5 days (H 2 O 2 group). To induce inflammatory stress, the cells were cultured in the growth medium supplemented with 10 ng/mL recombinant rat IL-1β (400-01B; PeproTech, Rocky Hill, NJ, USA) and TNF-α (400-14; PeproTech) for 5 days (IL/TNF group). The cells cultured in the growth medium were used as a control group.

SA-β-gal staining.
We performed SA-β-gal staining using a Senescence β-Galactosidase Staining Kit (#9860; Cell Signaling Technology, Danvers, MA, USA). Briefly, the cells were fixed with fixative solution for 10 min and incubated at 37 °C in staining solution at pH 6.0 for 16 h. SA-β-gal + cells were counted in four fields at × 10 magnification with a microscope (BZ-X700, KEYENCE, Osaka, Japan). ROS detection. ROS levels were evaluated using a ROS Assay Kit (R252; Dojindo, Kumamoto, Japan). The cells were plated in an 8-well 0.8 cm 2 Lab-Tek chamber slide (177445PK; Thermo Fisher Scientific). After 24 h, the cells were washed with phenol red-free Hanks' balanced salt solution (HBSS, 084-08965; Wako) and incubated in DCFH-DA solution and 0.5 μg/mL Hoechst 33342 (H342; Dojindo, Tokyo, Japan) for 30 min at 37 °C with 5% CO 2 . Thereafter, the cells were washed with HBSS and treated with H 2 O 2 and IL/TNF for 30 min. After washing with HBSS, the fluorescence signal was detected using a fluorescence microscope (BZ-X700, KEY-ENCE).
DNA and GAG quantification. The spheroids were digested with 100 µg/mL papain (P3125; Sigma-Aldrich) at 65 °C for 16 h. DNA content was determined with Hoechst 33258 dye (H341; Dojindo). Fluorescence intensity was measured with a microplate reader (Infinite M200; Tecan, Männedorf, Switzerland) at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Calf thymus DNA (D4522; Sigma-Aldrich) was used to generate a standard curve. GAG content was determined using a Blyscan Kit (B1000; Biocolor, Westbury, NY, USA) according to the manufacturer's instructions. The optical density at 656 nm was measured with a microplate reader. Finally, the total GAG content was also normalized to the total DNA content (GAG/DNA). H 2 O 2 and IL/TNF injection into rat knee joints. Six male Lewis rats aged 11 weeks old were used. All rats were housed in cages containing two rats each. They were given food and water added libitum under a 12 h light/dark cycle, and acclimatized for 1 week. Rats were given H 2 O 2 (8 mM, 50 μL) and IL/TNF (0.2 µg/mL, 50 µL) via an injection into their knees twice a week. These concentrations were set at 20 times the concentrations used in vitro. As a control, 50 μL of PBS was injected. After 4 weeks after the initial injection, the rats' knee joints were analyzed histologically.
Histology. Spheroids and knee joints were fixed in 10% neutral buffered formalin, embedded in paraffin, and sliced into 5-μm sections. The slides were stained with safranin O (1B463; Chroma Gesellschaft Schmid & Co., Munster, Germany) and fast green (061-00031; Wako) to visualize the distribution of GAG. Degeneration of the medial tibial cartilage was evaluated using the OARSI score 37  www.nature.com/scientificreports/ For p16, p21, MMP-13, ADAMTS-5, and IL-6, antigen retrieval was performed by immersing the sections in 10 mM Tris containing 1 mM EDTA (pH 9.0). For type I and II collagen, the sections were treated with 200 μg/ mL proteinase K (161-28701; Wako) for 10 min. For type II collagen, an additional step was carried out: 5 mg/ mL hyaluronidase (H3506; Sigma-Aldrich) was used for 1 h. Thereafter, the slides were blocked with Blocking One Histo (06349-64; Nacalai tesque, Kyoto, Japan) for 10 min and then incubated overnight with primary antibodies against p16 (